Clostridium difficile is the most common cause of nosocomial diarrhea in the United States. The disease has acquired greater significance in recent years because of rising case numbers and severity of disease, yet the virulence factors contributing to disease remain poorly understood. The gnotobiotic piglet has recently been developed as a model of C. difficile associated disease, including acute and chronic forms as seen in humans. The piglet model will be used in this project to further evaluate the specific roles of C. difficile Tcd/A and Tcd/B, the two key virulence factors. In Aim 1, a set of isogenic C. difficile toxin mutants will be created from the human epidemic 027/NAP1/BI strain. The mutants will be created using two recently developed methods for genetic manipulation in C. difficile, a task which, in the past, had been difficult. The isogenic mutants will allow comparison of disease severity when one or more toxins are not produced by the bacteria. In Aim 2, the piglet model will be used to analyze the relative role of TcdA and TcdB in disease in terms of local and systemic pathology and immune response. This will be accomplished by using a) the isogenic bacterial mutant strains produced in Aim 1, and b) through the neutralization of either toxin by the administration of hyperimmune polyclonal sera produced against each respective toxin in piglets orally challenged with the wild-type C. difficile strain. Accomplishment of the goals of this project will lead to greater understanding of the contribution of the two key toxins to C. difficile disease, which will help formulate the development of new treatment or prevention strategies in the future. Another significant outcome of this project will be the generation of a panel of isogenic mutant strains, and hyperimmune polyclonal sera against each of these two important toxins: useful tools for future investigations. PUBLIC HEALTH RELEVANCE: Clostridium difficile are bacteria that cause epidemic diseases throughout the United States and has become a major public health concern in health care facilities and the community. Understanding the ways in which C. difficile causes disease by describing the roles of its toxins will help generate new methods for prevention and treatment, as well a useful tools for future investigations.